This study aims to identify an entirely new class of caution transporter - the long-sought H+/Ca2+ exchanger - that operates within the Golgi complexes of mammalian cells and to determine its physiological role in the production of milk. The study is motivated by our recent discovery of a probable H+/Ca2+ exchanger within the Golgi complex of the budding yeast, the identification of TMEM165 as its sole ortholog in mammals, and the recent observations that TMEM165 is localized to the Golgi of mammalian cells and massively up-regulated in the milk-producing alveolar epithelial cells of the mammary gland uniquely at the time of milk production. The Golgi complexes of alveolar epithelial cells are the sites of both lactose biosynthesis and casein micelle formation, which together consume vast quantities of Ca2+ and generate large amounts of H+ as a byproduct. To test whether TMEM165 can transport H+ and Ca2+ in a coupled fashion, we propose to purify the protein in its native state from lactating mammary glands of cows, to reconstitute it into sealed liposomes, and to measure the rates of Ca2+ influx and efflux that occur in response to inward and outward pH gradients. We will test whether reconstituted TMEM165 can also promote H+ transport that is coupled to Ca2+ flux, and show that Na+ does not substitute for H+ or Ca2+ in these reactions. These experiments will define the first biochemical activities for the TMEM165-family of proteins. The physiological roles of TMEM165 in milk production will be tested using conditional knockout mice that specifically lack a critical TMEM165 exon in the mammary alveolar epithelial cells of late-pregnancy females. We expect the TMEM165-deficient Golgi complexes will exhibit a buildup of H+ that would inhibit transport of lactose precursors and alter the ability of caseins to complex with Ca2+ and form micelles, resulting in diminished milk production rate as well as alterations to its composition and nutritional quality. To test these predictions, we will measure lactose, casein, Ca2+, and H+ concentrations in milk of nursing TMEM165-deficient mice relative to control mice and we will the growth rates of litters nursing from TMEM165-deficient dams. These experiments will produce crucial biochemical and physiological support for the hypothesis that TMEM165-family proteins generally transport Ca2+ in and H+ out of acidic organelles. The experiments will help resolve the problem of milk de-acidification, and the results will provide insights into the functions of TMEM165 in other tissues and species, including the known cases of TMEM165-deficiency in humans. The findings will also broaden our general understanding of the normal mechanisms governing calcium homeostasis and signaling in all eukaryotes.